Eukaryotic organisms are made up of various cell types, each of which expresses a certain subset of specific genes. Since all cells basically inherit the same genetic information, there must be some regulatory mechanism which controls the expression of genes during differentiation. Recent experiments suggest that DNA methylation plays a role in this process since there is a correlation between gene activity and undermethylation in vivo and since enzymatic methylation inhibits the expression of several genes. The objective of this grant proposal is to understand the molecular modifications that occur during diffeentiation and to study the mechanisms by which methylation affects gene expression. These studies will be carried out using DNA mediated gene transfer as a means of introducing modified DNA into various cell types. Thus, by this technology, it will be possible to assay the effect of DNA methylation on specific and general protein-DNA interactions and the effect of DNA modification on transcription elongation, and on the process of DNA attachment to the nuclear matrix. In order to study the role of DNA modification during development, we will use differentiating rat myoblast cell lines and mouse erythroleukemia cells. We have also developed an elegant assay for the detection and isolation of factors which play a role in specific cell-type differentiation using RNA microinjection into tissue culture cells. One of the objects of this project will be to dissect out the role of DNA replication in the regulation of gene expression. Taken together, the results of these studies should shed light on the mechanisms of differentiation at the molecular level.